Orthogonal cloning of clpXP
- Get plasmid vectors and YFP-containing plasmid from Samantha. These are in E. coli that she has plated 14 July.
- Design primers based on plasmid vectors, restriction enzymes, and additional peptides needed at 5' ends.
- Get clpX, clpP genes in plasmids.
- Clone clpX, clpP, YFP, YFP+ssRAtag (8peptides+LAA) into yeast. Run PCR on plasmids containing these genes. Insert these genes into different yeast plasmid shuttle vectors (CEN/ARN and maybe 2 μm). Transform plasmids into yeast.
- Test for expression of clpX and clpP by Western blot.
- Test for functional protein degradation by clpXP. Determine if clpX is degrading proteins independent of clpP.
- Check if yeast is surviving the addition of clpXP. This may have come up after step 1.
- Measure rate of protein degradation via fluorescent signal of YFP.
- Troubleshoot in unlikely event that it doesn't work perfectly the first time.
- E. coli culture
- Constructing plasmid vectors
- Restriction enzyme use
- Cloning into plasmids
- Transforming yeasts
- Culturing yeast
- Western blot
- Fluorescence measurements
- Use Gal control of clpX, since it needs strongest control. Must have strong off (has worked for Samantha). Glucose would provide suppression in this system.
- Use Western rather than just fluorescence at start to make sure proteins are being expressed. Won't know if they are folding/working but would know if they're being expressed (and how much?). Antibodies exist in Sauer lab to all these proteins and to SSRA tag so you can follow any of these proteins.
[advanced techniques and troubleshooting options]
- Can't do polysystronic cloning in yeast, unless maybe using IRES (internal ribosomal entry site).
- ura-3 tag: can use dropout media, or introduce 5' FOA which creates a toxic byproduct, so positive and negative selection possible
- sterility factor